This disclosure relates to an improved analytical method which allows the relatively rapid release and direct analysis of analytes, including organic analytes, such as certain drugs of abuse or metabolites thereof, present in hair and other keratinized structures, e.g., fingernails and toenails. The method allows for the sensitive detection of such analytes without affecting the structure of the analytes and without being detrimental to analyte probes, e.g., antibody, RNA/DNA and bio-receptor probes, which may be used to detect the analyte. For example, in some embodiments, an analyte probe can be added directly to a keratinized structure which is suspected to contain one or more analytes and which has been treated as described herein. In this way, the identity of the one or more analytes as well as the extent and duration of consumption of the one or more analytes by a subject can be evaluated.
Analysis of hair and other keratinized structures has certain advantages over urine, blood, or oral fluid analysis techniques for the detection of analytes of interest. These include ease of handling and storage, a wide window of detection, and correlation of the presence and amount of drug with time of use and ingested dose. Urine, blood, and oral fluid techniques are known to be disadvantageous in that the duration and intensity of use or exposure cannot be ascertained. These techniques can, at best, provide short term information concerning ingested analytes. In addition, there are also problems with the interpretation of such results. For example, the detection of a low level of ingested drug or drug metabolite in the urine could mean that a subject ingested a small amount of the drug very recently or a larger amount several days earlier. Thus, chronic drug use typically cannot be determined with these methods without repeated testing.
In response to the problems of establishing a reliable and accurate method that would measure both the duration and intensity of analytes of interest, work performed by Dr. Werner A. Baumgartner, as reported in “Radioimmunoassay of Hair for Determining Opiate Abuse Histories”, J. Nucl Med 20:749-752 (1979), determined that long-term histories of exposure to drugs-of-abuse can be obtained through the analysis of mammalian body hair, since these substances are “trapped” within individual hair fibers during the synthesis of the fibers. In this respect, hair was shown to act like a tape recorder, i.e., past exposure histories can be evaluated through sectional analysis of hair samples. For example, it was found that morphine, once in the bloodstream, will find its way into hair as the hair is synthesized.
A variety of chemicals, including drugs-of-abuse, have been determined to be trapped by hair during its synthesis; these substances are “locked up” in hair for essentially the duration of the presence of the hair on the body. This was found to be true for head and body hair as well as for other keratinized structures such as fingernails; see Suzuki et al., Forensic Sci. International, 24:9-16, 1984. These entrapped substances cannot be washed out of hair, and were previously thought to be completely released only upon the complete, or nearly complete, destruction of the hair fiber.
Previous methods of extracting an analyte from hair included subjecting the hair to hot methanol solutions, or incubation of hair for hours (usually overnight) in an alkaline or acid medium; Yegles, et al., in: Analytical and Practical Aspects of Drug Testing in Hair, CRC Press, 2007, pp. 73-94; Jurado, C. in: Analytical and Practical Aspects of Drug Testing in Hair, CRC Press, 2007, pp. 95-125; Cheze, M. et al. in: Analytical and Practical Aspects of Drug Testing in Hair, CRC Press, 2007, pp. 163-185). Prior methods have also included the use of sonication or a mortar and pestle in conjunction with a solvent to aid in extraction.
Solvent extraction procedures can suffer from several problems in accurately determining the presence and amount of an ingested analyte. One of these problems is that the solvent extraction methods frequently remove only a small unknown and variable fraction of the total analyte present in the hair sample. Another disadvantage is that different analytes may require different solvents or different times and temperature for extraction. In addition, for analysis by immunoassay the solvents need to be evaporated, and many of the solvents are toxic and hazardous.
Other previous methods employed a combination of proteolytic and reductive treatments to completely digest and reduce the keratinized structures in order to release the one or more analytes. See, e.g., U.S. Pat. Nos. 5,466,579; 5,324,642; 6,022,693; 6,582,924; and 6,949,344, which are incorporated herein by reference, and which provide exemplary detection methods for both screening and confirmatory assays for analytes of interest, including immunoassay methods such as radioimmunassay and enzyme immunoassay methods. Such combined proteolytic and reductive treatment methods, while efficient, are relatively expensive due to the cost of the proteolytic enzyme, which can also interfere in subsequent analyte detection assays by proteolytically cleaving analyte detection probes such as antibodies, thereby preventing the use of certain highly sensitive analytical techniques or requiring the use of intermediate protease neutralization, separation, or purification steps.
Thus, there exists a need for an efficient and relatively inexpensive analyte detection method that can rapidly and completely release analytes from keratinized structures of the body such as hair, fingernails and toenails, and that can permit the direct determination of the identity of the analytes and their duration of use in a subject, without destroying or interfering with the analytes of interest and/or analyte detection probes such as immunoassay methods.